Specialized cell types allow plants to shed entire organ systems, such as leaves, flowers, and fruits. The ability to shed organs that have fulfilled their purpose enables plants to make efficient use of nutrients and energy sources. Abscission can also act as a plant defense mechanism: plants can protect themselves from disease by shedding infected organs. Organ shedding sometimes serves a propagative function, as in the seed dispersal promoted by fruit abscission.
Abscission zones are thought to differentiate as organs form, and can consist of a few to several cell layers of small, densely cytoplasmic cells. Prior to abscission, these cells have been shown to enlarge, and to secrete cell wall hydrolyzing enzymes such as cellulases and polygalacturonases. Secretion of polygalacturonase causes breakdown of the pectin-rich middle lamella between neighboring cells, thus allowing for cell separation to occur between abscission zone cells. After the organ has been shed, abscission zone cells left behind enlarge and form protective scar tissue (Bleecker and Patterson, 1997, Plant Cell 9: 1169–1179, which is incorporated by reference herein in its entirety).
Studies using abscission zone explants have demonstrated that ethylene promotes abscission (reviewed in Sexton and Roberts, 1982, Ann Rev Plant Physiol 33: 133–162; Osborne, 1989, Crit Rev Plant Sci 8:103–129, both of which are incorporated by reference herein in their entireties), and ethylene has been linked to abscission zone cell enlargement and zone-specific expression of hydrolytic enzymes (Jensen and Valdovinos, 1968, Planta 83: 303–313; Valdovinos and Jensen, 1968, Planta 83: 295–302; Wright and Osborne, 1974, Planta 120: 163; Koehler et al., 1996, Plant Mol Biol 31: 595–606; van Doom and Stead, 1997, J Exp Bot 48: 821–837, all of which are incorporated by reference herein in their entireties). A genetically defined role for ethylene response in the temporal regulation of abscission has been demonstrated by the discovery of ethylene-insensitive mutants such as etr1 and ein2 (Bleecker et al., 1988, Science 241: 1086–1089; Guzman and Ecker, 1990, Plant Cell 2: 513–523, both of which are incorporated by reference herein in their entireties). Studies of these Arabidopsis mutants have shown that ethylene insensitivity causes a delay in floral abscission, indicating that ethylene response mediates the timing of programmed cell separation (Bleecker and Patterson, 1997, Plant Cell 9: 1169–1179, which is incorporated by reference herein in its entirety). However, abscission does eventually occur in ethylene-insensitive mutants, albeit delayed, indicating that additional pathways must also regulate this process.
Although numerous studies have addressed hormonal regulation of the abscission process and physiological aspects of abscission zone cell separation, very few studies have focused on regulation of abscission zone development, with the result that the genes whose function is necessary for abscission to occur have not been identified in any plant system. Studies of genetic control of abscission zone development is being carried out using two tomato mutants known as jointless and jointless2 in which formation of pedicel abscission zones in tomato flowers is prevented; molecular characterization of the jointless locus is currently in progress (Butler, 1936, J Hered 27: 25–26; Wing et al., 1994, Mol Gen Genet 242: 681–688; Zhang et al., 1994, Mol Gen Genet 244: 613–621; Szymkowiak and Irish, 1999, Plant Cell 11: 159–176, all of which are incorporated by reference herein in their entireties).
Screens for abscission (abs) mutants in the model plant Arabidopsis have been carried out and several abs mutants have been isolated in which floral abscission is delayed. The lack of mutants for which organ abscission is specifically and completely blocked has limited the progress of studies of abscission zone development in Arabidopsis. 
The small GTP-binding protein ARF plays an established role in the control of vesicular traffic and in the regulation of phospholipase D (PLD) activity. GTPase activating proteins (GAPs) are associated with all families of small GTP binding proteins, acting as signal terminators and possibly also in some cases as effectors downstream of the GTP binding protein. The fact that ARF has undetectable intrinsic GTPase activity suggests that the ARF GAP is an essential terminator of ARF-regulated processes.
ARF has important roles in the control of vesicular traffic and in the regulation of phospholipase D activity. Replacement of bound GDP with GTP produces active ARF-GTP, which can associate with membranes. Both forms are important in vesicular transport, which requires that the ARF molecule cycle between active and inactive states. Like the many other GTP-binding proteins or GTPases that are molecular switches for the selection, amplification, timing, and delivery of signals from diverse sources, ARF finctions via differences in conformation that depend on whether GTP or GDP is bound. Vectorial signaling results from the necessary sequence of GTP binding, hydrolysis of bound GTP, and release of the GDP product (Moss & Vaughn, 1998 Jnl Biol Chem 273: 21431–21434, which is incorporated by reference herein in its entirety).
ARF proteins in their GTP-bound form are required for coatomer binding to Golgi stacks and for the binding of clathrin adaptor particles to the trans-Golgi network. GTP hydrolysis is required for the dissociation of these proteins from Golgi-derived membranes and vesicles, a process in which an ARF GAP is most likely involved, indicating that ARF GAPs are involved in vesicle coat disassembly as an uncoating factor.
Vesicular transport has been extensively studied in the Golgi and ER-to-Golgi pathways (Cosson & Letoumeur, 1997 Curr. Opin. Cell Biol. 9: 484–487, which is incorporated by reference herein in its entirety). The mechanisms, including the molecules and their functions, are likely very similar in other pathways. Formation of a transport vesicle begins when activated ARF with GTP bound to it associates with the cytoplasmic surface of a donor membrane. Activated ARF interacts with a coat protein, one of seven in the coatomer complex. Recruitment of multiple ARF molecules followed by coatomers causes membrane deformation and budding. Bilayer fusion at the base of a bud induced by fatty acyl-CoA results in vesicle release. Roles for PLD in both vesicle formation and fusion have been suggested. Removal of the coat, which is necessary for vesicle fusion at the target membrane, requires inactivation of ARF by hydrolysis of bound GTP to GDP.
Mammalian ARFs are divided into three classes based on size, amino acid sequence, gene structure, and phylogenetic analysis; ARF1, ARF2, and ARF3 are in class I, ARF4 and ARF5 are in class II, and ARF6 is in class III. Non-mammalian class I, II, and III ARFs have also been found. A role for class I ARFs 1 and 3 in ER to Golgi and intra-Golgi transport is well established (Cosson & Letourneur, 1997, Curr. Opin. Cell Biol. 9: 484–487, which is incorporated by reference herein in its entirety). ARF6 has been implicated in a pathway involving plasma membrane and a tubulovesicular compartment that is distinct from previously characterized endosomes (Radhakrishna & Donaldson, 1997, J Cell Biol 139: 49–61; Moss & Vaughn, 1998, Jnl Biol Chem 273: 21431–21434, both of which are incorporated by reference herein in their entireties).
An ARFI GAP (purified and cloned from liver) was recruited to membranes by overexpression of ERD2, a membrane receptor that recognizes the C-terminal sequence (Lys-Asp-Glu-Leu) found on certain soluble proteins (KDEL proteins) of the endoplasmic reticulum and serves to retrieve them if they are transported to the Golgi (Aoe et al., 1997, EMBO J. 16: 7305–7316, which is incorporated by reference herein in its entirety). Oligomerized ERD2 associated with the GAP, which then inactivated membrane-bound ARF and produced in the transfected cells a phenotype like that resulting from inhibition of ARF guanine-nucleotide exchange proteins (GEPs). It was later shown that overexpression of lysozyme with a KDEL terminus, which was intended to increase engagement of the KDEL receptor in retrograde retrieval transport, increased its interaction with ARF GAP and ARF inactivation, demonstrating a way in which vesicle content/cargo can influence a transport pathway (Cosson & Letourneur, 1997, Curr. Opin. Cell Biol. 9: 484–487, which is incorporated by reference herein in its entirety).
ARF GAP activity appears to be modulated by phospholipids. GAP activity is strongly stimulated by PIP2 and was inhibited by phosphatidylcholine, as indicated by Makler et al (1995, Jnl Biol Chem 270: 5232–5237, which is incorporated by reference herein in its entirety) using both crude and purified GAP preparations. The effects of phospholipids on the ARF GAP may be related to a recently discovered role of ARF in the regulation of phospholipid metabolism (Kahn et al., 1993, Cell 75: 1045–1048, which is incorporated by reference herein in its entirety), where ARF was identified as the cytosolic GTP binding protein that activates phospholipase D. Activated phospholipase D cleaves phosphatidylcholine to produce phosphatidic acid and choline. A feedback loop mechanism has been proposed where following the activation of phospholipase D by GTP-bound ARF, an increase in local phosphatidic acid concentration (and possibly also a decrease in phosphatidylcholine concentration) brings about an increase in the activity of the ARF GAP, resulting in the hydrolysis of ARF-bound GTP and the cessation of phospholipase D activity.
In other experiments using recombinant GAP, dioleylglycerol dramatically increased the activity of the recombinant GAP (amino acids 1–257). Because monosaturated diacylglycerols are produced chiefly from PC via the sequential action of PLD and phosphatidate phosphohydrolase (whereas polyunsaturated diacylglycerols are derived from PIP2 via phosphatidylinositol phospholipase C action), it was suggested that PLD activity could be a major regulator of ARF GAP (Antonny et al., 1997, J Biol Chem 272: 30848–30851, which is incorporated by reference herein in its entirety). GAP activity was varied 100-fold by altering relative amounts of PC and diacylglycerol (Antonny et al., 1997, supra, which is incorporated by reference herein in its entirety), and similar effects were observed on the activity of and lipid binding by Gcs1, an analogous ARF GAP from yeast.
By comparing systematically the effects of phospholipid polar head groups and hydrocarbon chains on binding to the two GAPs, it was concluded that membrane association depended chiefly on hydrophobic interaction of the protein with hydrocarbon moieties of the lipid, which is favored by small head groups, and the conformation of monounsaturated acyl chains (Antonnye et al., 1997, supra, which is incorporated by reference herein in its entirety). In this view, the activation of ARF GAP results from increasing its concentration at the membrane where ARF-GTP resides. ARF activation of PLD leading to decreased PC and increased diacylglycerol levels would promote translocation of ARF GAP to a vesicle membrane where it could inactivate ARF-GTP and thereby terminate PLD action. The ARF GAPs that are activated by PIP2 or other phosphoinositides are presumably subject to different kinds of regulation.
The process of abscission in plants affects many important physiological events in the various stages of plant growth. Thus, the ability to control abscission would be beneficial for many aspects of plant biology and crop science. What is needed in the art is a method of genetically modulating the process of organ abscission in plants.